CN102026699B - Micro-device and method for non-invasive and selective separation and extraction of particles in polydispersed suspensions, production method, and applications thereof - Google Patents

Micro-device and method for non-invasive and selective separation and extraction of particles in polydispersed suspensions, production method, and applications thereof Download PDF

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CN102026699B
CN102026699B CN2008801261783A CN200880126178A CN102026699B CN 102026699 B CN102026699 B CN 102026699B CN 2008801261783 A CN2008801261783 A CN 2008801261783A CN 200880126178 A CN200880126178 A CN 200880126178A CN 102026699 B CN102026699 B CN 102026699B
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characterized
cell
micro device
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CN102026699A (en
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马里亚·伊西亚尔·冈萨雷斯戈麦斯
托马斯·戈麦斯阿尔瓦雷斯-阿雷纳斯
路易斯·乔斯·费尔南德斯莱德斯马
阿尔弗雷多·卡拉托梅纳
乔斯·路易斯·索托马丁内斯
哈维尔·贝甘佐鲁伊斯
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康斯乔最高科学研究公司
米格尔·埃尔南德斯大学
埃尔切市大学总医院基金会
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Priority to ES200703248A priority patent/ES2326109B1/en
Application filed by 康斯乔最高科学研究公司, 米格尔·埃尔南德斯大学, 埃尔切市大学总医院基金会 filed Critical 康斯乔最高科学研究公司
Priority to PCT/ES2008/070230 priority patent/WO2009071733A1/en
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B03SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03BSEPARATING SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS
    • B03B5/00Washing granular, powdered or lumpy materials; Wet separating
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/08Detecting organic movements or changes, e.g. tumours, cysts, swellings
    • A61B8/0833Detecting organic movements or changes, e.g. tumours, cysts, swellings involving detecting or locating foreign bodies or organic structures
    • A61B8/085Detecting organic movements or changes, e.g. tumours, cysts, swellings involving detecting or locating foreign bodies or organic structures for locating body or organic structures, e.g. tumours, calculi, blood vessels, nodules
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/44Constructional features of the ultrasonic, sonic or infrasonic diagnostic device
    • A61B8/4444Constructional features of the ultrasonic, sonic or infrasonic diagnostic device related to the probe
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/48Diagnostic techniques
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34Filtering material out of the blood by passing it through a membrane, i.e. haemofiltration, diafiltration
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61MDEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3678Separation of cells using wave pressure; Manipulation of individual corpuscles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D21/00Separation of suspended solid particles from liquids by sedimentation
    • B01D21/28Mechanical auxiliary equipment for acceleration of sedimentation, e.g. by vibrators or the like
    • B01D21/283Settling tanks provided with vibrators
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D43/00Separating particles from liquids, or liquids from solids, otherwise than by sedimentation or filtration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • B01J8/16Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes with particles being subjected to vibrations or pulsations
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04Cell isolation or sorting
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02Separating microorganisms from their culture media
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781Aspects relating to microreactors
    • B01J2219/00925Irradiation
    • B01J2219/00932Sonic or ultrasonic vibrations
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B29WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29CSHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C65/00Joining or sealing of preformed parts, e.g. welding of plastics materials; Apparatus therefor
    • B29C65/02Joining or sealing of preformed parts, e.g. welding of plastics materials; Apparatus therefor by heating, with or without pressure
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B29WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29CSHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C66/00General aspects of processes or apparatus for joining preformed parts
    • B29C66/01General aspects dealing with the joint area or with the area to be joined
    • B29C66/02Preparation of the material, in the area to be joined, prior to joining or welding
    • B29C66/028Non-mechanical surface pre-treatments, i.e. by flame treatment, electric discharge treatment, plasma treatment, wave energy or particle radiation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B29WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29CSHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C66/00General aspects of processes or apparatus for joining preformed parts
    • B29C66/50General aspects of joining tubular articles; General aspects of joining long products, i.e. bars or profiled elements; General aspects of joining single elements to tubular articles, hollow articles or bars; General aspects of joining several hollow-preforms to form hollow or tubular articles
    • B29C66/51Joining tubular articles, profiled elements or bars; Joining single elements to tubular articles, hollow articles or bars; Joining several hollow-preforms to form hollow or tubular articles
    • B29C66/53Joining single elements to tubular articles, hollow articles or bars
    • B29C66/534Joining single elements to open ends of tubular or hollow articles or to the ends of bars
    • B29C66/5346Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat
    • B29C66/53461Joining single elements to open ends of tubular or hollow articles or to the ends of bars said single elements being substantially flat joining substantially flat covers and/or substantially flat bottoms to open ends of container bodies
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B29WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29LINDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
    • B29L2031/00Other particular articles
    • B29L2031/756Microarticles, nanoarticles
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10TTECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T156/00Adhesive bonding and miscellaneous chemical manufacture
    • Y10T156/10Methods of surface bonding and/or assembly therefor
    • Y10T156/1002Methods of surface bonding and/or assembly therefor with permanent bending or reshaping or surface deformation of self sustaining lamina
    • Y10T156/1039Surface deformation only of sandwich or lamina [e.g., embossed panels]

Abstract

The invention relates to a micro-device for non-invasive and selective separation and extraction of particles in polydispersed suspensions by means of the strategic use of ultrasounds, laminar flow and standing wave effects in a channel produced in a chip by means of microtechnology. Said device is a resonant multi-layer system comprising a modified ''lambda quarter wavelength'' treatment channel, which enables the particles to be channelled and separated in a flow inside the substrate channel without touching the walls of the device, in order to avoid problems of adherence. Said micro-device can be used in the field of biomedicine and/or biotechnology for the separation and concentration of cells, preferably human cells, that can be used for investigative and medical methods for diagnosis and treatment.

Description

For selective and non-invasively micro device and method, manufacture method and the application thereof of the particle of separation and extraction polydispersion suspension

Technical field

The present invention relates to a kind of device, its in conjunction with microtechnology and ultrasonic wave as for Selective Separation with extract the method for the particle of polydispersion suspension, described polydispersion suspension contains the microcomponent that all has different physical properties (size, density or compressibility) for any concentration level, and described device is mainly applicable to biomedicine and biological technical field.

Background technology

In the past in 10 years, in different technology is applied especially the field such as biotechnology and pharmacy, the different technologies for the suspended particulate matter of handling or separating has been proposed.At present, the separation of particle is especially paid close attention to and the medical application of donating blood, dialysis procedure is relevant with lab analysis, and recirculation and/or the cleaning of the rear blood of operation.

To suspension, apply sound standing wave and produce the effect that some equilibrium area that the node set up to the standing wave to producing distributes relevant with maximum sound pressure power value transmits particle in medium.The main radiant force that acousta induction is sent out is applied each particle, and its size changes pro rata with operating frequency.Distance the reducing along with node and maximum pressure value that the particle that stands this power is advanced for arriving nearest acoustic balance position.These limit by wavelength, and itself and acoustic frequency are inversely proportional to.Therefore, from view of theory, easier enrichment particle under higher frequency.

This Noninvasive transport mechanism is known in ultrasonic field, and in the past decade, has proposed the trial development of several manipulations and/or separating particles.Proposed with the different technologies of this phenomenon from separating particles liquid or other fluids.Usually, flow is crossed conduit or the passage that wherein is formed with the sound standing wave vertical with passage length.As a result, particle moving is to form and to concentrate band along popin weighing apparatus position in these conduits.

Nearest patent WO 2007/044642 A2, " DEVICE AND METHOD FOR COMBINED MICROFLUIDIC-MICROMAGNETIC SEPARATION OF MATERIAL IN CONTINUOUS FLOW " (Ingber Donald, Xia Shannon, Tom Hunt, Peter Westervelt) open continuous-flow cell separation apparatus based on magnetic field application.Operation principle is from of the present invention fully different the application of electromagnetic energy because it is not based on mechanical energy, and with the present invention by contrast, it is invasive technique, needs to insert in the inorganic particles of pending suspension outside.These easily particulate affected by magnetic fields adhere to some cell in suspension and pull them, they are transferred to the equilibrium area of pre-selected with magnetic force.Owing to being invasive method, therefore after medium, extracting, the viability of the cell separated is partly changed for follow-up study.Two characteristics of this of this device make this patent very different from device of the present invention, although the two uses simultaneously and have the double-fluidized-bed of an interface under laminar region.

J.Statis.Mech. paper: " Theory and experiments; 2006; " Continuous particle size separation and se size sorting using ultrasound in a microchannel " (Sergey Kapishnikov; Vasily Kantsler; Victor Steingberg) discloses two kinds by the ultrasonic device that particle in suspension is separated and classifies with cell.Wherein the first is so-called " λ/2 resonators " (λ is wave length of sound), wherein set up the standing wave that has pressure node in passage central authorities, particle is to its transmission with concentrate in the sound field application process, and they continue to flow until arrive outlet along it.By this way, enrichment the suspension of ultrasonic concentrated particle by central flow pass separating device, the remaining suspension owner will be by two other with respect to the symmetrically arranged side of centre gangway flow pass separating device simultaneously.In device, it is impossible in whole passage, being uniformly distributed that the remaining ingredient that exists in mobile suspension definitely separates with particle that acoustics is collected.Owing to not affected by ultrasonic field, their space layout does not change in the sound field application process, and they are through three flow pass separating devices, although they are mainly to leave by lateral access by this way.This is because ultrasonic concentrated cell leaves centre gangway together.In order to realize Selective Separation (author is called " cell classification "), article also proposes to develop λ/4 resonators, wherein channel width is approximately 1/4th of wavelength, and to its conveying and collect selected cell one of lateral access wall near the build-up pressure node.With the present invention by contrast, this device need to be used two ultrasonic transducers, it is placed in the both sides for the treatment of channel cross section, outside and parallel to each other opposed at passage, emission has the poor interim wave train of variable phase.This device is near device of the present invention, but their marked difference is that it can cause some performance constraint (depending on the circumstances), if do not applied continuous ripple and restriction district (this is determined with respect to how much symmetric configurations of central treatment channel by the space retrained between two ultrasonic wave emission sources and inflow and flow pass) physically, thus strong restrictions the ul-trasonic irradiation district.Opposite with the present invention, this λ/4 devices are approximately moving under f=4MHz, and to processing volume (its cross section and frequency are inversely proportional to (approximately little 4 times than the present invention)) with because there is remarkable restriction the required channel measurement accuracy aspect of aforementioned reason.

Patent WO/2002/072236 PARTICLE SEPARATION (ERYSAVE AB, Henrik, LAURELL Thomas, ALLERS Mats, PERSSON Hans) disclose a kind of utilization and be used to form a plurality of particles and concentrate a plurality of acoustic pressure nodes of band to separate the device of the particulate matter (being particularly useful for blood processing) in resonator.It is a kind of device that a great difference is arranged in design and operating aspect and the present invention.Sound field be applied to from λ/2 or the very different process chamber of λ/4 resonator passages on, this process chamber has than they larger sizes can set up the standing wave with a plurality of nodal spatial distributions and maximum sound pressure value.

European patent EP 1365849, also be published as U.S. Pat US6929750, US2004069717 and WO02072235 " DEVICE AND METHOD FOR SEPARATION ", (LAURELL Thomas, ALLERS Mats, Henrik, PERSSON Hans) disclose a kind of microfluidic device, it separates for the ultrasonic wave in the half-wave resonator that forms the spatial distribution structure that is called as " array " of be arrangeding in parallel.Transportation and sound wave collection that cell is entreated wherein, wherein occur in the resonator passage of these elements each self-contained being called " λ/2 ".As in such other resonators, when finishing, process obtains the suspension of the acoustic effect cell of enrichment; But it does not form leaching process to remaining suspension, but by the collection with high concentration level of central flow pass acquisition.This device is used on the expensive silicon substrate in the microdevice of this type and implements in customization.This patent imagination increases the Acoustic treatment amount by a plurality of ultrasonication passages that be arranged in parallel.These composed components that form the mount board of technical being called " array " comprise single entrance and three flow pass of introducing suspension separately, two symmetrically arranged lateral access that the suspension of preconcentration collecting granules (red blood cell) therefrom flows through through the center-aisle of its discharge and all the other suspensions.The component of the power impact of those not rayings in sample, keeps being uniformly distributed and through any one separating device in three flow pass, described three flow pass comprise that (although degree is lower) red blood cell is all by the center-aisle of its discharge.

Patent WO 00/04978CONCENTRATION OF PARTICLES IN A FLUID WITHIN AN ACOUSTIC STANDING WAVE FIELD, (CEFAI Joseph BARROW; David Anthony; COAKLEY William Terence; HAWKES Jeremy John) a kind of device for the suspended particulate matter of handling fluid is disclosed.It comprises the conduit that suspension flows through, and for setting up ultrasonic transducer and the reflector of the standing wave vertical with the flow direction of passage.This device is " λ/2 resonators ", and it forms the single particle band in conduit central authorities.Distance between transducer and reflector is less than 300 microns, corresponding to process chamber rather than resonant frequency ultrasonic transducer.According to the structure tolerance of device and the character of pending suspension, device has been imagined the change programme to this distance and processing frequency.Collect suspended particle and by the acoustic pressure node to the port transmission that goes out that is placed in the device lateral access, the suspension of enrichment particle is through exporting thus separating device.Simultaneously, other particles are via another flow pass separating device that is placed in another direction.

WO 98/50133 (WO 1998/050133) PARTICLE MANIPULATION, (COAKLEY William Terence; HAWKES Jeremy John, BARROW David Anthony CEFAI Joseph) a kind of device of handling suspended particulate matter is disclosed, it consists of the conduit with a plurality of sizes that equal the half-wavelength specific factor, suspension flows via it, and sets up the standing wave vertical with flow direction therein.In this process chamber, set up standing wave with a plurality of nodes and maximum sound pressure value with form acoustics that collect, parallel, at a distance of half-wavelength with perpendicular to the cell band of circulating direction.

At IBM, Technical Disclosure Bulletin Vol.25, No.1, disclose a kind of ultrasonic Continuous Flow plasma separator in June 1982,192/193.This device is comprised of the transducer of two quadrature settings, and each has reflector and processing volume therebetween, so that the suspension of dilution stands standing acoustic waves.This process chamber is from of the present invention very different, although the two all takes form and the process chamber changed.

JP 06241977 A discloses a kind of device of measuring fine particle by the ultrasonic standing wave method, in conjunction with electrostatic force acoustics, causes to concentrate the separating particles with separation different size by supersonic beam.Central authorities produce pressure node at process chamber, and particle is to this node transmission and concentrated.

EP0773055A2 (1996) (YASUDA K, SAITAMA H G, UMEMURA S, HUDRIOHI S) interaction that discloses a kind of supersonic beam produced based on several transducers of being placed by different tactics comes acoustics to handle the apparatus and method of particle.Suspension stands the acoustic levitation process by the ultrasonic field of the interaction generation of several bundles of a plurality of supersonic sources generations by being set to directly and/or indirectly contacting with medium in chamber.

The device that WO 93/19367A2 discloses a kind of acoustics method of agglomeration and consisted of pipe, described pipe comprises fluid sample and connected ultrasonic transducer, to produce the standing wave perpendicular to pipe, thus particle is concentrated in a plurality of transverse belts, described particle is along described a plurality of transverse belt sedimentations after applications of ultrasound.It is based on U.S. Patent No. 5,665, and 605 and 5,912,182.

JP 07 047259A discloses a kind of for delivery of the short grained device in suspension.Described device comprises a plurality of ultrasonic wave producing components on two flat surfaces that are arranged on the two-dimensional transducer panel; Pending liquid deposition therebetween.

Summary of the invention

One aspect of the invention is a kind of for the selective and micro device of the particle of separation and extraction polydispersion suspension non-invasively, micro device of the present invention hereinafter referred to as is characterised in that it comprises the following assembly in the chip substrate that is integrated in the acoustics soft material:

A) flow micro-channels system, it has the asymmetric spatial distribution of the flow pass branch that comes from central treatment channel, and it comprises:

I. initial suspension is along its path of flowing or bed, and it comprises for the flow channel of supplying with and leave the flow pass of described device through it, parallel with ii,

Ii. the pure fluid parallel with i is along its path of flowing or bed (being called the fluid collection bed), and from described pure fluid, extracting selected particle, described path or bed comprise inflow or feed path and flow pass, form iii

Iii. central treatment channel, in this place, stating initial suspension and pure fluid separates by the boundary interface of the streamline under the laminar region being limited by the passage lateral dimension, described central treatment channel is divided into two flow pass in its path ends, the width of wherein said passage (being inversely proportional to acoustic frequency) is slightly larger than 1/4th of wave length of sound, and the degree of depth (250 μ m) that forms this treatment channel of channel cross-section together with described width is less than 1/4th of wave length of sound basically

With

B) ultrasonic actuator or the transducer on one of sidewall,

-described ultrasonic actuator or transducer are also in parallel outside central treatment channel, it is to the chip that forms multilayer system-combination of channels part emission acoustic energy, the resonance of described multilayer system correctly is arranged in described passage respectively apart from the about distance of 1/3 and 2/3 channel width of reflector walls pressure node, and generation sound causes barometric gradient in centre gangway, it has the effect that specific particle is wherein carried to the phono-equalizer unit district that is defined in node, radiant force is invalid herein

-its sphere of action not only comprises described treatment channel, also comprises the bifurcation region that flows out microchannel towards two, and purpose is that Selective Separation and extraction efficiency are maximized.

A particular of the present invention is micro device of the present invention, the constituent material of wherein said chip substrate is preferably epoxy resin SU-8, realize described passage thereon, the acoustic impedance of acrylic compounds PMMA (polymethyl methacrylate) substrate is 3.3MRayls, and ultrasonic transducer b wherein) can be small-sized piezoelectric pottery or rectangle piezoelectric composite, preferably 1-3 level piezoelectric composite.

Another aspect of the invention is the manufacture method of micro device of the present invention, hereinafter referred to the manufacture method of micro device of the present invention, based on the photoetching technique according to the described design of Fig. 1, it comprises the following steps:

A) polymeric layer that deposition and restriction light can limit on individual substrate (wafer 1) surface,

B) be coated with on the individual substrate of non-adhesive material (wafer 2) surface deposition and limiting the polymer SU-8 layer that light can limit,

C) sealing wafer 1 and wafer 2, be disposed opposite to each other the side that contains the polymeric layer that light can limit of described wafer, and

D) remove the wafer covered by non-adhesive material.

Therefore, another aspect of the present invention is the purposes of micro device of the present invention, hereinafter referred to purposes of the present invention, for the selective process of separating, washing and/or classifying with Noninvasive of the particle of polydispersion suspension.

The present invention another more specifically aspect be purposes of the present invention, the cell that wherein said particle belongs to following (only for not limiting the scope of the invention for example): virus, prion, prokaryotic (bacterium, saccharomycete, fungi, algae etc.) and eukaryotic.

A purposes that more particular embodiment is micro device of the present invention of the present invention, it is for Selective Separation and isolation eukaryotic, preferred human body cell, more preferably tumour cell, haemocyte (red blood cell, blood platelet, macrophage and lymphocyte), stem cell or mother cell, no matter be whether in body cell or embryonic cell or body fluid such as blood, urine, celiolymph, exist or from other kind cells that exist in the other types biological sample in tissue biopsy.

Developed for by having, having the multi-layered type ultrasound resonance of the improvement of single characteristic λ/4 passages device to carry out ultrasonic Separation and extract the particle of suspension and the micro device of cell.Particularly, about the inflow of central treatment channel and sample and the flow pass asymmetric spatial distribution aspect with respect to central treatment channel, it has specific geometric configuration.The present invention also finds the importance of this last characteristic, and it has strengthened separative efficiency, as mentioned below.

The present invention is based on the following fact: the inventor observes by the application of processing the shock wave that the transducer of channel parallel produces with central authorities and produces the standing wave perpendicular to flow direction, and its pressure node is placed in passage central authorities and the centre position that occupies between the reflector walls in the shared zone of pure liquid bed of the length that is subjected to acoustics effect treatment channel is located.

This device is excited by ultrasonic actuator or transducer, for example small rectangular piezoelectric ceramics or have the 1-3 level piezo-electricity composite material of low-down surface vibration amplitude (be less than in end 10% and actual in centre be zero).The piezoelectric fabric of described piezo-electricity composite material in being embedded in polymer nature forms with configuration example such as 1-3 level composite material.Like this, and the coupling between the transverse mode of its Size dependence with and through chip, minimize to passage and inner emission.

One of the present invention is characterised in that supersonic source is set to locate to contact with chip in one of outer rim, parallel with treatment channel, on the vertical direction for the treatment of channel length, passes its thickness emission acoustic energy.Piezoelectric element part on one of its metalized surface is glued to one of external margin of micro device chip, especially the most closely is arranged on chip and the edge of central treatment channel in parallel.Like this, its emission acoustic energy passes the pantostrat that forms multilayer system, in treatment channel, sets up the standing wave perpendicular to flow direction with pressure node.

Treatment channel is important with respect to the position of device assembly, but is not conclusive; Particularly, the inventor has developed two kinds of chip configurations, and it has two kinds of distances from the passage to the chip edge, and wherein ultrasonic actuator is placed on chip edge to improve pressure node stability, wherein:

The-the first configuration, the distance between treatment channel and ultrasonic actuator is 750 μ m (are equivalent to wavelength 3/8);

The-the second configuration, the distance between treatment channel and ultrasonic actuator is 500 μ m (are equivalent to wavelength 1/4).

Two kinds of devices have all obtained effective separation and extraction process, but in the second configuration, can be observed some optimization of passage interior nodes stability.In the resonance of multilayer system, at each assembly (composite-chip-passage-chip), participate in the foundation of passage interior nodes.

Therefore, the standing wave that the pantostrat by device produces is redistributed according to the first configuration, has optimized the position of the pressure node in passage, and acoustic energy that can optimization device.Different from this device, considering that resonance forms in passage and exterior layer does not make a strong impact, so most of technological development is symmetrical, do not allow this possibility.The configuration diversity certain degree of the application's multilayer system increases the running parameter for possible technological improvement.

Acoustic pressure gradient on the sidewall of passage, be placed in around the node in the centre position between treatment channel central authorities and reflector walls, formed by the zone that pure fluid bed occupies in the whole length of acoustic effect passage.This pressure node is respectively apart from about 1/3 channel width and producing apart from the distance of wall 2/3 channel width on opposite of emission wall.Therefore, the pressure distribution of setting up on the direction vertical with sample flow in whole passage length is derived from the radiant force that acts on each suspended particulate on the direction perpendicular to flow direction with ad hoc fashion.But its laser propagation effect only limits to have the acoustic condition impact that is subject to apply and certain size, density or constrictive those particles of selecting according to the particular type applied in all cases, described particle accelerates under the effect of described power.

The strategic location that forms the acoustic balance district of the pressure node in pure liquid bed makes the particle that acoustics pulls cross two separating interfaces between fluid media (medium), thereby leave the suspension (hereinafter also referred to as collecting fluid) that will be collected in pure fluid, they extract through one of flow pass from here.

The inventor also points out the importance of facility strategy location (far away apart from reflector walls), because its representative is for the wherein innovation of " λ/4 resonators " of the close reflector walls of node.This prevents by particle and adheres to described wall and the problem that causes, and is beneficial to it and flows to concentrating of channel outlet, thereby avoids obstructing problem.In addition, pressure node occupies and the similar length along passage of ultrasonic actuator (length that piezoelectric ceramics or 1-3 level piezo-electricity composite material occupy).

Another innovative point of the present invention relates to the inflow that comes from central treatment channel and the asymmetric layout of flow pass, and it makes the ultrasonic wave conducting system by chip exerting one's influence on the wider aisle spare of the isolator than being subjected to common these types (comprising branch) effect.This tactic space layout has improved the possibility of arranging supersonic source (the gummed zone of piezoelectric ceramics or piezo-electricity composite material) in chip substrate, and increases the active region in treatment channel, comprises the zone of flowing out branch's bifurcated to two.(different from the remainder geometry of passage), be conducted to channel outlet between the gained radiant force in this zone, from this particle, collects the fluid separating device, to improve the efficiency of Selective Separation.

The expansion of sound active region guarantees that selected particle flows out through the passage of expectation, and making it is the efficiency optimization of separation and extraction suspension from starting medium.

Of the present invention another is characterised in that ultrasonic processing frequency is 1MHz, is less than usually this class micro device frequency used (usually in minimum 2MHz work, most of higher than this value).But this frequency can change, thus the lateral dimension of the central treatment channel of convergent-divergent (must with wavelength produces be varied to direct ratio (being inversely proportional to acoustic frequency)) easily.Therefore, low frequency allows to process larger processing volume.But in the situation that frequency is less than 500kHz, acoustic cavitation threshold value (its generation by the microvesicle that has strong and quick implosion effect in medium forms) is also lower than the acoustic cavitation threshold value of high frequency.Therefore, produce ultrasound-transmissive and the microactuator suspension component do not caused to the acoustic energy excursion of damage is more limited.In addition, must consider that radiant force increases with acoustic frequency is linear, due to this point, the volume benefit of using resonator to report at low frequency has the shortcoming of high energy consumption.In the situation of micro device of the present invention, ultrasonic in the situation that use lower than common frequency, specifically at 1MHz, but the acoustic cavitation threshold value is high, its feasibility is described and allows to process volume to be at least for 2 times in the device situation of 2MHz resonance.Therefore, the present invention provides two advantages about this parameters,acoustic with respect to the existing resonator micro device under upper frequency; These relate to the increase of aforementioned processing volume and the therefore minimizing of the restriction relevant with measuring adjustment precision (being essentially conduit wall).

Another innovative point of micro device of the present invention is its composition material, by the composition base substrate (thickness be about 900 μ ms) of the integrated chip of two parallel connecting materials: PMMA (polymethyl methacrylate) for channel bottom, the thin layer of the epoxy resin SU-8 that can limit with the light be arranged on described substrate (thickness is 330 μ m), be provided with passage on it.In this, special reflecting device etc. not yet is used to the wall of central treatment channel, and on the contrary, the superperformance of SU-8 material is firmly established, and it forms the major part of resonance multilayer system in device.Its low acoustic impedance allows the coupling of its resonance mode and passage, and need to be for the treatment of special reflecting device of conduit wall etc.Therefore, polymeric material SU-8 as reflector element for the favourable applicability of setting up the purposes of standing wave and these acrylic compounds acoustics soft materials in passage by experiment confirm.In addition, the good acoustics behavior of the constituent material PMMA of channel bottom is verified is the transmitter with ultrasonic energy of the acoustic characteristic of similar SU-8 and good machinery-acoustics coupling.They are two kinds and are easy to process and polymeric material cheaply.Bi-material all has low acoustic impedance (not higher than 3 times of water with at least than low five times of metal) and can be easy to process to produce passage, and with this class micro device in the obvious advantage of the cost as lower as silicon of other substrates used, the latter sees more rigidity and more expensive from acoustic angle.In a word, they provide attractive economic advantages.

The model for experiment of describing in the second practical embodiment is that the ps particle by different size and density forms, it can for example simulate physics and the acoustic characteristic of following two types of cells: red blood cell and the tumour cell come off from peripheral blood, originally it flow together in being similar to the fluid of blood plasma, and any other contains other samples of the microcomponent of these characteristics.

It is worth mentioning that in the Selective Separation of the particle with maximum gauge that utilizes device of the present invention to obtain in being implemented in experiment and the high efficiency of extraction.The repetition behavior is to be caused by the independent role of acoustic radiation power to each particle, and the concentration in suspension is irrelevant with them.This effect is all effectively for high concentration and extremely rare suspension, and other isolation technics are showing sharply and reducing aspect effect sensitivity and efficiency.

In a word, in all above-mentioned innovative points, the simplicity of micro device and validity are very outstanding: simplicity comes from the geometric configuration of supersonic source (consisting of piezoelectric ceramics or piezo-electricity composite material) and treatment channel and inflow and outflow branch; And the constituent material of chip of device: the plastic material SU-8 (passage is set on it) on the PMMA substrate that forms the channel base substrate, with and effective result.

Therefore, one aspect of the present invention is a kind of micro device of the particle for selective and Noninvasive separation and extraction polydispersion suspension, micro device of the present invention hereinafter referred to as is characterized in that it comprises the following assembly in the chip substrate that is integrated in the acoustics soft material:

A) flow micro-channels system, it has the asymmetric spatial distribution of the flow pass branch that comes from central treatment channel, and it comprises:

I. initial suspension is along its path of flowing or bed, and it comprises for the flow channel of supplying with and leave the flow pass of described device through it, parallel with ii,

Ii. the pure fluid parallel with i is along its path of flowing or bed (being called the fluid collection bed), and from described pure fluid, extracting selected particle, described path or bed comprise inflow or feed path and the flow pass that forms iii,

Iii. central treatment channel, in this place, stating initial suspension and pure fluid separates by the boundary interface of the streamline under the laminar region being limited by the passage lateral dimension, described central treatment channel is divided into two flow pass in its path ends, the width of wherein said passage (being inversely proportional to acoustic frequency) is a bit larger tham 1/4th of wavelength, and the degree of depth (250 μ m) that wherein forms the treatment channel of cross section together with width is less than 1/4th of wave length of sound basically

With

B) ultrasonic actuator on one of sidewall or ultrasonic transducer,

-also in parallel outside central treatment channel, it is to the chip that forms multilayer system-combination of channels part emission acoustic energy, the resonance of described multilayer system correctly is arranged in described passage respectively apart from launching the approximately distance of 1/3 and 2/3 channel width of wall pressure node, with be created in the barometric gradient produced on the inner acoustics of centre gangway, it has the effect that the acoustic balance district that the radiant force limited in node in it is invalid carries some particle, its sphere of action not only comprises central treatment channel, also comprise the zone of flowing out the microchannel bifurcated to two, purpose is that Selective Separation and extraction efficiency are maximized.

In the present invention, use " particle " in term " polydispersion suspension " to refer to have different physical characteristics the are arranged suspension of particle of (size, density or compressibility etc.), comprise inorganic or organic micro-component such as cell, preferred eukaryotic, more preferably human cell, the micro-component of other types of the same levels parameter occurred in microorganism or biofluid.

Use term " chip that the acoustics soft material is made " to refer to that impedance is far below other materials or medium such as metal or glass (low five times at least), basically be no more than the material of three times (usually being limited in variable range, except exception generally changes between 0.8MRayls~2.6MRayls) of liquid medium impedance.Therefore, concept " soft " refers to the impedance relationship between the fluid of the constituent material for the treatment of channel wall and its internal flow, sets up standing wave but have the reflection that enough capacity produce sound wave.

Therefore, any soft material with the acoustic characteristic that is similar to SU-8 or other plastic components, preferred acrylic material, all can be used as on it material of the chip substrate of the manufacture micro device of the present invention that passage is set, its reason is it at the similitude aspect acoustic energy propagates and similar reflex response on conduit wall.

A specific embodiments of the present invention is micro device of the present invention, constituent material (preferred epoxy SU-8) and acrylic compounds substrate PMMA (polymethyl methacrylate) that the chip substrate of passage wherein is set on it have the acoustic impedance of 3.3MRayls, and ultrasonic transducer b wherein) can be small-sized piezoelectric pottery or piezo-electricity composite material, preferred 1-3 level.

Another aspect of the present invention is the manufacture method of micro device of the present invention, and hereinafter referred to the manufacture method of micro device of the present invention, based on the photoetching technique according to the described design of Fig. 1, it comprises the following steps:

A) polymeric layer that deposition and restriction light can limit on individual substrate (wafer 1) surface,

B) be coated with on the individual substrate of non-adhesive material (wafer 2) surface deposition and limiting the polymer SU-8 layer that light can limit,

C) sealing wafer 1 and wafer 2, be disposed opposite to each other the side of containing the polymeric layer that light can limit of described wafer, and

D) remove the wafer covered by non-adhesive material.

Those skilled in the art can easily produce this micro device by knowledge and the current state of design and this area that the present invention provides.In addition, the design of micro device of the present invention can strengthen by introducing extra empty passage, and described extra empty passage strategy is arranged on around centre gangway so that pass through the minimization of loss of the acoustic energy of PMMA chip substrate and the emission of SU-8 material.C) and d) and below centre gangway, add two sealing filling channels, these extra elements can easily be incorporated in the design of apparatus of the present invention in parallel therewith the step b of the manufacture process by duplicating device).Below the fluid treatment passage that separation occurs and near it, filling channel is set.Like this, for separating of ultrasonic signal be placed in the restriction site place of expectation, thereby make minimization of loss.The configuration of centre gangway also can be by following reinforcement:

-change the position with respect to the micro device sub-assembly, to strengthen the stability of channel inner pressure node.Particularly, by the distance between the outer wall that changes passage and chip near ultrasonic actuator, described distance can reduce to 1/4 (the acoustic energy propagates maximum under the frequency of selecting) at the most of described medium medium wavelength.Therefore, the stability that is conducive to channel inner pressure node (collecting micro-component to its transmission and acoustics).

-treatment channel is reduced by half, and dwindle whole device with identical ratio.Shortening reduces their interface in the distance that the treatment channel internal mutual parallels two mobile sample processes with same ratio.Therefore, diffusion path length obviously shortens.In fact, non-newtonian fluid as for example blood (its viscosity with some parameter change) in, the shortening of passage is to avoiding interface to break and preventing that it from invading whole passage is necessary.On the other hand, the shortening of passage means fluid shorter time of staying and therefore still less Acoustic treatment therein, and larger controllability.

In addition, the operation of micro device can strengthen by adjusting a little operating frequency, because system shows WD microoperation ability, makes and can around the core work frequency designed for it, produce slight frequency change.According to application leaved for development, increase and allow to change equilbrium position and towards the micro-component in the position collection channel of expectation according to application leaved for development lower than the frequency of its core values 12%.This characteristic is given micro device and is applied widely diversity.

On the other hand, the operation of micro device can strengthen by widening operating frequency range, and this is because make system have micro-maneuvering capability by the very little variation of frequency around the core work frequency around for design.According to application leaved for development, make the collection of micro-component in equilbrium position and passage change to desired location lower than the frequency increase of its core values 12%.This characteristic is applied diversity widely to micro device.

On the other hand, micro device of the present invention also the available heat stamping technology in conjunction with follow-up gummed process, with following method manufacture:

A) prepare the mould comprising the expection channels designs,

B) the molded substrate that will use of mould of gained in using a) under pressure and/or temperature action, and

C) in the effect of pressure and/or temperature and/or oxygen plasma surface active, by with another plastic material gummed seal substrate.

On the other hand, for micro device of the present invention can with frequency range for organic and inorganic suspension, be all very wide, but in organic suspended matter, to consider some factor of the following stated.A change programme of ultrasonic frequency means the convergent-divergent process of the size of device.While considering the operation of micro device, based on the acoustic resonator model on channel width direction, the spatial character relevant to this lateral dimension of process microchannel must change inversely with acoustic frequency.Although the radiant force that the micro-component of each of suspension produces directly is directly proportional to frequency, in the situation of the organic suspended matter with low frequency level (kHz magnitude), must considers the reduction of sound cavitation energy threshold value, thereby avoid primary cellular defect.Low frequency is favourable to this phenomenon of not expecting, therefore to the present invention, will be restricted in the application lower than 500kHz.On the contrary, frequency increases the linear amplitude that increases radiant force, and make produce selective ultrasonic be that the main required acoustic energy levels of transmission descends.For this reason, nearly all device of exploitation is worked between 2MHz~5MHz at present.On the contrary, the raising of these frequencies means that the ratio of the lateral dimensions for the treatment of channel reduces, and this must change pro rata with the variation that sound wave causes, due to the needs of accuracy, has improved the cost of the manufacture process of these devices.

By the result that this model obtains, make application of installation in particle separation and isolation field, important application is agricultural biotechnologies, due to separation and the isolation as for example preferred human cell of cell of the biotechnology of human and animal's health, core diagnosis and therapeutic process, for example cell or the preferred human diseases of gene therapy mammal disease.

Therefore, another aspect of the invention is the purposes of micro device of the present invention, hereinafter referred to purposes of the present invention, for selectively with Noninvasive, separating, wash and/or the process of the particle of the polydispersion suspension of classifying.

The present invention another more specifically aspect be purposes of the present invention, wherein particle comprises the cell (only not limiting the scope of the invention with example) that belongs to following: virus, prion, prokaryotic (bacterium etc.) and eukaryotic.

More specifically embodiment of the present invention is micro device of the present invention for Selective Separation and isolation eukaryotic (as algae, fungi, comprise saccharomycete), preferred human body cell, no matter whether more preferably tumour cell, haemocyte, stem cell or mother cell be in body fluid such as blood, urine, celiolymph or the cell of the body cell, embryonic cell or the other types that exist in the biological sample of the other types in tissue biopsy.

No matter whether relevant to diagnosis or treatment, the spendable concrete bioprocess of micro device of the present invention or application relate to donate blood, plasmapheresis, dialysis procedure and lab analysis, and circulation and/or the cleaning of blood after operation, the cell that wherein needs some type for example red blood cell separates and concentrates with hematoblastic.

Another example is that micro device of the present invention is applied to mankind's diagnosis and/or therapeutic process, and for Selective Separation and extraction patient's damage or the cell of change, it can and be reused for the patient in external reparation.

A specific field of biologic applications is oncology, can be used as diagnosis and the instrument of omen, for to having solid tumor that different tissues originates from and at the circulating tumor cell (CTC) of the peripheral blood of the tumor patient in various disease stage, carrying out the reproduction of Selective Separation and extraction.

Clinical use show the quantification in the quantity of the circulating tumor cell of peripheral blood concentrate on up to now following aspect:

● the independence of breast cancer and metastatic prostate cancer is examined factor (Cristofanilli M in advance, BuddGT, Ellis MJ, Stopeck A, Matera J, Millar MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF.Circulating tumour cell, disease progression and survival in metastatic breast cancer N Engl J Med 351:8, 2004) (Moreno JG, Milelr MC, Gross S, Allard WJ, Gomella LG, Terstappen LW.Circulating tumour cell predict survivalin patients with metastatic prostate cancer.Urology, 65 (4): 713-718, 2005).

● monitoring has reaction (the Cristofanilli M of the tumor patient of terminal illness to chemotherapy, Mendelsohn J, et al.Circulating tumour cell in breast cancer:advanced tools for tailored therapy.Proc Natl Acad Sci USA 103 (46): 17073-17074; 2006).

Analytical system used is based on the positive immunity of monoclonal antibody and separates and subsequent analysis use fluorescence microscope in these researchs.The U.S. these the application obtained Food and Drug Administration (FDA) to it usage license in clinical practice.

Two kinds of application are day by day extensive for the application of the tumour of other types.Equally, there is Primary Study to show potential application (the Soto JL of CTC number analysis as the early stage label to the colorectal cancer recurrence, Garrigos N, Gallego J, Guaraz P, Garcia-Bautista M, Castillejo A, Gomez A, Casado-Llavona C, Rodriguez-Lescure A, Carrato A.Toward a circulating tumour cell analysis as an early marker for relapse in stage II and III colorectal cancer patients.Eur J Cancer Supplements.3 (2): 187, 2005).

One of main advantage of device of the present invention is effective separation of C TC (and be easy to its counting) and can separate described cell mass to carry out the realistic possibility of subsequent analysis under feasible condition, two kinds of descriptions are all in gene level and gene expression scope, and the research of external behaviour.At present, this is unique known device that high efficiency this possibility like this can be provided.

The concept of CTC has following added value as affordable Noninvasive tumor biopsy: can the described cell of functional sign at them with respect to the performance aspect the sensitiveness/tolerance of existing methods for the treatment of, as personalization system, be used to each patient the most effectively treatment now.

Therefore the reality and potentiality purposes of this device is extremely important for clinical practice, for the various disease for the treatment of better the patient provides of great value information.

The inventor also finds the importance of this last characteristic, and it has strengthened separating effect, as detailed below.

The accompanying drawing explanation

Fig. 1 illustrates wherein micro device effect of the present invention and the element of transportation suspended particulate matter and the perspective view (2D) of mode.

Fig. 2 illustrates the multilayer configuration of device of the present invention.

Chip is shown Fig. 3 and piezoelectric ceramic transducer is integrated in the photo for the device prototype on the assembling sheet that inserts/extract fluid.

Chip is shown Fig. 4 and piezoelectric ceramic transducer is integrated in the photo for the device prototype on the assembling sheet that inserts/extract fluid.

Fig. 5 illustrates photo and the figure of PMMA chip.

Fig. 6 illustrates the microphotograph of channel interior.

Fig. 7 illustrates the microphotograph of each 20 μ m particle to the behavior of displacement separately of the acoustic pressure node in passage.

Fig. 8 illustrates the photo of 20 μ m particles.

Fig. 9 illustrates the photo of particle separation/extraction.

Figure 10 illustrates the leaching process of 20 μ m particles through flow pass.

Figure 11 illustrate use up can limit (fotodefinible) material as the figure of the manufacture method of the micro device of the present invention of structural material.

Figure 12 illustrates the design of micro device of the present invention, wherein at its end part seal the passage of filling air, strategically arranges with respect to centre gangway, so that the Minimal energy loss in seeing through the emission process of substrate.A) chip of observing from above, b) cross section of chip.

Figure 13 illustrates the figure by the manufacture method of drop stamping technology manufacture micro device of the present invention.

Figure 14 illustrates micro device and applies the active effect of sound and control ability in the micro-component in passage, by the minor variations of frequency core values, make the modification of equilbrium position and the collection (930kHz) of the micro-component in passage to desired locations, reach reflector walls (1.1MHz) according to application leaved for development.

Specific embodiments

The first embodiment has been described the first prototype of micro device of the present invention.

In prototype shown in Figure 4, be used to the structural material of realizing fluid passage, be the polymer SU-8 that light can limit, its mechanical bond is to the PMMA substrate, the channel bottom of this PMMA substrate constituent apparatus.Due to the possibility [ref4] that the up rightness [ref2] of its high restriction (micro-meter scale), the wall that grows, bio-compatibility [ref3], wide thickness range [ref1] and glue connect several pantostrats, this material has characteristic very easily to manufacturing installation.According to the design of Fig. 1, the manufacturing step of this prototype of apparatus of the present invention used following (seeing Figure 10):

A) the poly-compound layer that deposition and restriction light can limit on the surface of individual substrate (wafer 1),

B) the polymer SU-8 layer that deposition and restriction light can limit on the surface that is coated with the individual substrate of non-adhesive material (wafer 2),

C) sealing wafer 1 and wafer 2, make on the described wafer side containing the polymeric layer that light can limit positioned opposite to each other, and

D) remove the wafer covered by non-adhesive material.

The prototype of micro device comprises with being fabricated to: the chip (100) with integrated system of four microchannels (160,162,170 and 180), take central treatment channel (110) and be central authorities in described four microchannels, every end has two, be asymmetric setting, for inflow and the outflow (seeing Fig. 1,2 and 3) of the two media of PARALLEL FLOW under the layer-shaped area along passage (110).One of these media are therefrom to extract the suspension (150) of (101) specific particle, and another medium is the neat liquid (124) as the ultrasonic collector of particle (101).

As seen from Figure 2, the ultrasonic transducer (190) that is glued to one of metalized surface on chip edge comprises two kinds of polymeric materials (a kind of formation passage, the substrate of another kind of formation and its mechanical connection), and the generation ultrasonic wave, described ultrasonic wave is delivered to treatment channel through the acrylic compounds chip.Channel width " w " is about 1/4th of wave length of sound, and allows to set up standing wave on the described direction in passage, and described passage has pressure node in the distance of the approximately w/3 with respect to an one sidewall, as reflector.

Photo 3.a shows the device of observing from top, and photo 3.b illustrates the chip structure that has two kinds of mechanical connection polymeric materials along limit.

Visible in photo as Fig. 4, device has ultrasonic actuator or the transducer be integrated in one side (190), and it can (have 1 by small rectangular PZ26 piezoelectric ceramics

The thickness mode of MHz resonates) or 1-3 level piezo-electricity composite material, it by the part of metalized surface, is glued to the thickness of the chip consisted of SU-8 and the part by lateral edges is glued to the PMMA substrate, and part occupies its thickness and is parallel to the treatment channel setting.Supersonic source is set to contact with Chip Vertical.To perpendicular direction emission ultrasonic energy, its mode is carried out on the direction perpendicular to flow direction for the medium emission acoustic energy in passage.

More specifically, channel width be 390 ± 4.6 μ m (1.06 be multiplied by 1MHz wavelength 1/4).It is 117 ± 4.6 μ m that pressure node is set to apart from the distance of reflector walls, in the zone occupied by pure liquid bed, outside suspension.Therefore, passage has 0.0975mm 2Cross-sectional area and the wavelength that can freely change, but in concrete situation of the present invention, be 1cm.Therefore, channel volume is 0.975mm 3.

After Figure 10 is illustrated in the single ultrasound-transmissive under the extremely low flowing velocity of about 0.06mm/s, extract particle through the flow pass on the right, it allows visual and quantize the acoustics behavior clearly.The clear validity that ultrasonic selection separating treatment is shown of continuous photogram (10.a)~(10.d), wherein diameter is all particle separating devices of 20 μ m and moving to pure catcher fluid flow pass, the remaining suspension thing that contains simultaneously 6 little μ m particles flows along its left side " bed ", by corresponding passage discharge.

Figure 11 illustrate use up can limit the figure of manufacture method of the micro device of the present invention manufactured as structural material of material.

A) on the surface of individual substrate deposition and limit that light can limit polymeric layer; B) on the surface of the individual substrate that is coated with non-adhesive material deposition and limit that light can limit polymeric layer; C) sealing wafer 1 and wafer 2; And d) remove the wafer covered by non-adhesive material.

Figure 12 illustrates the design of micro device of the present invention, wherein has the filling channel of hermetic terminal with respect to the setting of passage strategy, so that see through Minimal energy loss in the emission process of substrate.A) chip of observing from above, b) cross section of chip.

Figure 13 illustrates the figure that manufactures the manufacture method of micro device of the present invention by the drop stamping technology.

A) preparation is comprising the mould of the design of expectation passage, b) the molded substrate that will use of mould of gained in utilizing a) under pressure and/or temperature action, and c) under the effect of the surface active of pressure and/or temperature and/or oxygen plasma, carry out seal substrate by being glued to another plastic material.

Figure 14 illustrates the minor variations of micro device by frequency values in the active effect of its acoustics to the micro-component in passage and control ability, and the minor variations of this frequency values allows to change equilbrium position and collect upper infinitesimal (930kHz) to the passage of reflector walls (1.1MHz) towards desired location according to application leaved for development.

In the second embodiment, the purposes of micro device of the present invention in the particle of separation simulation cell described.

For the microfluidic control of micro device of the present invention, use to have to be applied to simultaneously three different volumes (the constant voltage injection pump of the ability of syringe of 10 μ l~110ml) is used for controlling each entrance (160 and 162) and locates flowing of two media.Suspension (150) and catcher fluid (124) are passed through respectively these entrances (160 and 162) with the syringe of equal volume (each 5ml) and are injected simultaneously under uniform pressure.

In catcher fluid (124) bed, utilization monitors in real time the ultrasonic Separation of selected particle by the photograph of the microscopic resolution of CCD camera and to the transmission of pressure node, this CCD camera is connected to the optical lens sub-assembly of the resolution ratio with 1.17 μ m/ digital pixels.The width of the liquid bed that suspension occupies remains on 1/2 left and right of passage (110) width.

Research vehicle in the present embodiment is polystyrene microparticle model, it has the physics of the following two kinds of cells of simulation and size and the density of acoustic characteristic: red blood cell and the tumour cell come off from peripheral blood start to flow together in being similar to the fluid of blood plasma.

The physics of this fluid and acoustic characteristic, for example its density and sound propagation velocity are at document (Cousins CM, Holownia P, Hawkes JJ, Limaye MS, Price CP, Keay P, Coakley WT, Plasma preparation from whole blood using ultrasound.Ultrasound Med Biol 26:881-888, 2000) in, describe, and erythrocytic these characteristics (Duck F A, Physical properties of tissue:a comprehensive referente book, Academia London, 1990, Haider L, Snabre P, Boynard M, Rheology and ultrasound scattering from aggregated red cell suspensions in shear flow, Biophysical Journal, Vol.87, 2322-2334, 2004).On the contrary, do not exist the bibliography that makes tumour cell flows in peripheral blood these characteristics to make us by exploitation, test guidance model and indirectly determine described data.For this reason, we are by means of the two kinds of acoustic characteristic that often is applied to be undertaken by centrifugal and density gradient in the cancer laboratory two kinds of liquid mediums of cell separation: Ficoll Technical undefined but be used to being called Oncoquick with another kind The liquid medium of experimental provision, the latter is very effective in separating tumor cell due to its density, floating thereon [the Rosenberg R of tumour cell come off from peripheral blood, Gertler R, Friedrichs J, Fuehrer K, Dahm M, Phelps R, Trovan S, Nekarda H, Siewert JR.Comparision of two density gradient centrifugation systems for the enrichment of disseminated tumour cells in blood.Cytometry 49; 150-158.2002].

The possible variable amplitude of TCD derives from two kinds of density and sound propagation velocity measured value: 1.030gr/cm in liquid 3<ρ (tumour cell)<1.055gr/cm 3, the uncertainty of minimum of a value is less than 5%.Based on these data and consider the approximately linear dependence of biological micro-component, selecting in this embodiment density is 1.05gr/cm 3Particle as the representative of tumour cell, estimate their compressibility.

After utilizing this experimental model sign tumour cell, simulate them with the granules of polystyrene that selected diameter is 20 μ m.Although the variable range of these cells very wide (can be defined as 10~40 μ m), select this size as standard value.

In this mode, we further introduce and analyze the aqueous suspension thing of " two dispersion ", and the diameter that namely comprises variable concentrations is respectively two kinds of particle swarms of 6 and 20 microns, and in device, carries out ultrasonic to it.Selective Separation and the oarse-grained result of extraction can be known and see in Fig. 6,7,8 and 9.

According to Fig. 6, can be observed the distribution of two media in passage: when there is no ultrasonication, suspension along the left side of passage, flow and water on right side.The larger particles that some diameters that suspension mainly flows by the little granules of polystyrene of 6 micron diameters of high concentration with low-down concentration (Cv<1%) are 20 microns forms.At Fig. 6 .a) photo in, we can be observed the cross direction profiles of medium under low flowing velocity, make the particulate fraction of 6 μ m visible.Photo resolution is 1.17 μ m/ pixels.On the contrary, corresponding to Fig. 6 .b of higher flowing velocity) photo do not obtain described differentiation, but shown the different tones of brightness contrast between two media.

According to Fig. 7, the particle that is positioned at passage top is affected by ultrasonic actuator and stand lateral displacement on perpendicular to flow direction at the starting point place of channel part, intensity is less than the particle that is positioned at the photo bottom, and the latter is subjected to sound field affect and pulled more consumingly fully.Therefore, described particle is positioned at the position of pressure node, and does not also reach this position at the time course middle and upper part particle that obtains photo.

Fig. 8 illustrates the picture corresponding to the photo of two kind of 20 μ m particle, and described two kind of 20 μ m particle flows very lentamente along passage in pressure node, be positioned at the acoustic pressure Nodes, separates from the emission wall.

Fig. 9 illustrates the experimental result of photograph of the separated/extracted process of the selected particle undertaken by flow pass.What the first photogram (9.a) showed is that 20 μ m particles left side branch by passage together with remaining suspension does not flow out naturally along the fluid bed that it flows when there is no ultrasonic wave.Photogram (9.b), (9.c) and (9.d) show the selective outflow of these particles, with suspension, separate, by pure fluid-water, flow out passage, after pressure node transmission and acoustics collection, be positioned at (right half part) on pure liquid bed, and continue to flow until leave treatment channel along it.All these photograms are corresponding to identical film.In these series, in whole passage, the flowing velocity of particle is 2.4mm/s.

For (excursion injects at 0.06mm/s~1.4mm/s) test that all samples of (always below the required layer-shaped area of micro-fluidic technologies) carries out, and the result that the selection of 20 μ m particles separates is all positive with different in flow rate by central treatment channel.Use two aqueous suspension things that disperse: have the non-quantized granule (6 μ m) of different volumes concentration, and the diameter of concentration very low (generally lower than 1%) is the bulky grain of 20 μ m.All tests are all carried out under the determined frequency by channel width, this frequency is strategically determined based on the PZ26 piezoelectric ceramics resonant frequency of 1MHz.Always use deionized water as pure fluid.

More specifically, by two passages (160 and 162) by the parallel introducing centre gangway of two media: suspension (150) (has the particle (101) of particular characteristics, especially has 20 μ m diameter and 1.05gr/cm from its extraction 3The particle of density), and liquid fluid (deionized water) (124), described two passages have same cross-sectional area (0.049mm 2) and be integrated in chip of the present invention, each passage occupies half of centre gangway (110) separately.Two media flows through abreast passage (110) and flows through along the layer-shaped area below of their corresponding beds: pure fluid will be along liquid bed (124) collecting granules (101), and suspension occupies other parts in passage (110) cross section along bed (122), thereby stablize at the interface (120) that keeps separating them.The behavior can be observed in the photo of continuous two flow velocitys (being respectively 0.06mm/s and 1.4mm/s) of Fig. 5.Photo 5.a illustrates the microchannel of enforcement and is glued to the position of the piezoelectric ceramics of chip edge.Fig. 5 .b illustrates the cross-sectional view of the chip consisted of two polymeric material SU-8 and PMMA.After the length that covers passage (110), two media divides to two flow pass (170 and 180) at bifurcation (175), and from this separating device.When suspension (150) flows along the bed (122) in passage (110), the foundation of the standing wave produced in passage (110) due to the PZT (piezoelectric transducer) by outer setting (190), those particles (101) of the certain size wherein comprised and density stand radiant force.

Service voltage due to 15 volts that apply from from the continuous signal generator to ultrasonic transducer, 20 μ m particle (101) experience radiant forces, and under ul-trasonic irradiation along passage (110) with the direction of the flow of suspension perpendicular to continuous to the pressure node fast transport, be positioned at the shared zone (Fig. 6) of pure fluid (water) (124), they continue to channel end mobile (Fig. 7) through this zone, in the mode of differentiation, through being immersed in flow pass (180) separating device in described fluid (124), and before using ultrasound, comprise their suspension and separate.

On the contrary, in suspension (107), 6 μ m granules of contained high concentration are not affected by sound field and acoustics do not occur to pull, its reason is that radiant force is directly proportional to the cube of radius, because its radius ratio bulky grain (101) is little three times, therefore much smaller to its radiant force applied.Like this, the fluid bed that starts along them in suspension of particle continues to flow and deflection path not.Finally, they flow out the passage separating device through suspension.

Fig. 8 and 9 illustrates two groups of continuous photograms, wherein can be observed 20 μ m particles in the ultrasonic extraction of the suspension from them and being collected in catcher fluid after to the bifurcation of centre gangway, flow, they by passage (180) separating device, separate with their starting medium therefrom.1/4 of the wave length of sound of being slightly larger than for the width of routing (110) to set up therein, the place, centre position between reflector walls and interface (120), namely the position of the 1/3-2/3 of respective channel (110) forms the acoustic pressure node approximately respectively.Along in the channel flow process, from moment of active region that they enter sound field, particle (101) tends to concentrate on the node location of standing wave at particle (101).

Remaining suspension component (150) is not affected by sound field, do not cross the interface (120) between two media (150 and 130), but continue to flow, corresponding bed (122) along it in whole microfluidic channel (110) flows, until arrive bifurcation (175), they are by flow pass branch (170) separating device from this.

It should be noted that the Selective Separation obtained and extract the oarse-grained height validity of 20 μ m in utilizing the experimentation of apparatus of the present invention.

For example, in sample is injected to treatment channel with the 5ml syringe with the flowing velocity of 1.4mm/s (12 minutes injection 1ml) in passage experiment, do not find to produce detrimental effect, wherein 20 μ m bulky grains flow out the passage separating device by suspension, rather than continue transmission towards catcher fluid, they are discharged by this flow pass.

The visual quantitative analysis of carrying out to the sample in two passages (170) and the collection of (180) exit has confirmed from Selective Separation the suspension that ultrasonic processing is immersed 20 μ m bulky grains before and has extracted the oarse-grained validity of 20 μ m.The liquid of collecting 1 minute from the passage of the suspension that stands sound wave through its discharge does not contain 20 μ m particles, but finds to exist 6 very many μ m granules.On the contrary, the liquid of collecting in the exit of passage (180) contains 20 μ m granules of polystyrene, if can in centre gangway (110) and in stub area (175), observed before, from its initial suspension through the acoustics separation and extraction to catcher fluid (124), by passage (180) separating device.

These experiments are in the situation that 20 μ m granule densities very low (much smaller than 1% volumetric concentration) carry out, and have simulated the truth of tumour cell from coming off blood.In this particle swarm of variable concentrations (always being less than 10%), found the repetition behavior, its reason is and can be regarded as the independent role that is applied to the acoustic radiation force on each particle, and the concentration in suspension is irrelevant with it.Effect the Usefulness Pair high concentration effective with extremely rare suspension, other isolation technics are the sharply decline of display sensitivity and validity.

Claims (21)

1. one kind for the selective and micro device of the particle of separation and extraction polydispersion suspension non-invasively, it is characterized in that it comprises the following assembly in the chip substrate that is integrated in the acoustics soft material:
A) flow micro-channels system, it has the asymmetric spatial distribution of the flow pass branch that comes from central treatment channel (110), and it comprises:
I. initial suspension is along its path of flowing or bed, and it comprises for the flow channel (160) of supplying with and leave the flow pass (170) of described device through it,
Ii. the pure fluid (124) parallel with i is the fluid collection bed along its path of flowing or bed, from described pure fluid (124), extracting selected particle, described path or bed comprise inflow or feed path (162) and flow pass (180), form iii
Iii. central treatment channel (110), in this place, stating initial suspension and pure fluid separates by the boundary interface of the streamline under the laminar region being limited by the passage lateral dimension, described central treatment channel (110) is divided into two flow pass (170,180) at its path (175) end, the width of wherein said central treatment channel (110) is less than 1/4th of wave length of sound basically, form its cross section
With
B) ultrasonic actuator or the transducer on one of sidewall (190),
-described ultrasonic actuator or transducer are also parallel with it outside central treatment channel (110), it is to the chip that forms multilayer system-combination of channels part emission acoustic energy, the resonance of described multilayer system makes pressure node correctly be arranged in described central treatment channel respectively the distance apart from reflector walls 1/3 and 2/3 central treatment channel (110) width, its sphere of action comprises described central treatment channel (110) and towards two bifurcation regions that flow out microchannel, purpose is the maximizing efficiency that makes Selective Separation and extraction.
2. according to the micro device of claim 1, it is characterized in that the size of described central treatment channel (110) width is greater than 1/4 of wave length of sound.
3. according to the micro device of claim 1, it is characterized in that the size of described central treatment channel (110) degree of depth is less than 1/4 of wave length of sound.
4. according to the micro device of claim 1, the acoustics soft material that it is characterized in that described chip substrate is the material of three times that its impedance is no more than the liquid medium impedance, and the variable range of described impedance is 0.8MRayls~2.6MRayls.
5. according to the micro device of claim 4, it is characterized in that the acoustics soft material of described chip substrate is acrylic material.
6. according to the micro device of claim 1, it is characterized in that described acrylic material is epoxy resin SU-8.
7. according to the micro device of claim 1, it is characterized in that described PZT (piezoelectric transducer) is piezoelectric ceramics or piezo-electricity composite material.
8. according to the micro device of claim 7, it is characterized in that described piezo-electricity composite material is the 1-3 level.
9. method of utilizing the drop stamping technology to manufacture the micro device of any one in claim 1 to 8 is characterized in that it comprises the following steps:
A) in individual substrate, be on wafer 1 surface, to deposit and limit the polymeric layer that light can limit,
B) in the individual substrate that is coated with non-adhesive material, be on wafer 2 surfaces, to deposit and limit the polymer SU-8 layer that light can limit,
C) sealing wafer 1 and wafer 2, be disposed opposite to each other the side of containing the polymeric material that light can limit of described wafer, and
D) remove the described wafer covered by non-adhesive material.
10. method of utilizing the drop stamping technology and manufacturing the micro device of any one in claim 1 to 8 in conjunction with follow-up gummed process is characterized in that it comprises the following steps:
A) prepare the mould comprising the expectation channels designs,
B) the molded substrate that will use of described mould obtained in using a) under pressure and/or temperature action, and
C) under the effect of pressure and/or temperature and/or oxygen plasma surface active, by with another plastic material, gluing together to seal described substrate.
11. the purposes of any one micro device required for protection in claim 1 to 8, in the process of selectively with non-invasively separating, wash and/or classifying of its particle for the polydispersion suspension.
12., according to the purposes of claim 11, it is characterized in that described particle is selected from:
-virus,
-prion, and
-cell.
13., according to the purposes of claim 12, it is characterized in that described cell is prokaryotic.
14., according to the purposes of claim 13, it is characterized in that described prokaryotic is bacterium.
15., according to the purposes of claim 12, it is characterized in that described cell is eukaryotic.
16., according to the purposes of claim 15, it is characterized in that described eukaryotic is selected from:
-fungi,
-algae, and
-human cell.
17., according to the purposes of claim 16, it is characterized in that described human cell is selected from:
-tumour cell,
-haemocyte,
-stem cell, and
-mother cell.
18., according to the purposes of claim 17, it is characterized in that described mother cell is body cell.
19., according to the purposes of claim 17, it is characterized in that described mother cell is embryonic cell.
20., according to the purposes of claim 16, it is characterized in that described fungi is saccharomycete.
21. the purposes of the desired micro device of any one in claim 1 to 8, is characterized in that the eukaryotic separation process is for blood, plasma exchange process, dialysis procedure and lab analysis.
CN2008801261783A 2007-12-05 2008-12-05 Micro-device and method for non-invasive and selective separation and extraction of particles in polydispersed suspensions, production method, and applications thereof CN102026699B (en)

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